Load

Libraries and functions

Warning message in rsqlite_fetch(res@ptr, n = n):
“Don't need to call dbFetch() for statements, only for queries”
==========================================================================
*
*  Package WGCNA 1.63 loaded.
*
*    Important note: It appears that your system supports multi-threading,
*    but it is not enabled within WGCNA in R. 
*    To allow multi-threading within WGCNA with all available cores, use 
*
*          allowWGCNAThreads()
*
*    within R. Use disableWGCNAThreads() to disable threading if necessary.
*    Alternatively, set the following environment variable on your system:
*
*          ALLOW_WGCNA_THREADS=<number_of_processors>
*
*    for example 
*
*          ALLOW_WGCNA_THREADS=4
*
*    To set the environment variable in linux bash shell, type 
*
*           export ALLOW_WGCNA_THREADS=4
*
*     before running R. Other operating systems or shells will
*     have a similar command to achieve the same aim.
*
==========================================================================


Allowing multi-threading with up to 4 threads.
[1] "preparing gene to GO mapping data..."
[1] "preparing IC data..."
[1] "preparing gene to GO mapping data..."
[1] "preparing IC data..."
[1] "preparing gene to GO mapping data..."
[1] "preparing IC data..."

Data

GF_104w_F_1_2GF_104w_F_2_2GF_104w_F_3_2GF_104w_M_1_2GF_104w_M_2_2GF_52w_M_1_2GF_52w_M_2_2GF_52w_M_3_2GF_52w_M_4_2GF_8w_M_1_2SPF_8w_F_3_2SPF_8w_F_4_2GF_8w_F_1_2GF_8w_F_2_2GF_8w_F_3_2GF_8w_F_4_2GF_8w_F_5_2GF_104w_M_3_2GF_104w_M_5_2GF_104w_M_4_2
0610005C13Rik 0.00000 0.00000 0.00000 1.762244 1.255776 0.8428813 4.933784 5.600068 7.223366 8.467973 2.104621 2.598973 18.22456 0.0000 2.02195 5.263542 0.0000 1.256186 1.048314 1.510221
0610007P14Rik 863.76231 747.10939 907.37337 882.884096 990.178987 509.1002974529.888431 514.086266 737.505715 603.645538 741.878817 907.041617 897.29135 681.3924 611.63981 592.674855 807.4643 494.937435 597.539204 749.069592
0610009B22Rik1015.87139 1165.23596 1177.13303 1220.353766 1554.022190 1040.1155082875.253330 1015.852381 876.916688 948.413030 1160.698348 1294.288611 1038.79966 1019.3717 1140.37968 959.017393 1091.5484 692.158697 809.298711 788.335337
0610009L18Rik 28.06775 36.08199 18.16563 20.265803 38.301153 32.0294889 26.642435 22.400273 4.334020 61.695235 45.249346 102.659438 48.24147 19.5615 11.12072 45.266463 24.3135 25.123728 23.062917 39.265745
0610009O20Rik 216.39328 278.04355 265.21824 225.567194 271.247510 300.0657382349.311927 395.364819 309.160084 360.493728 251.502181 227.410148 231.55906 158.6655 270.94127 304.232741 268.7282 337.914137 367.958352 397.188110
0610010F05Rik 409.24587 459.51473 287.92528 277.553384 303.269785 392.7826798405.557067 388.644737 357.556640 343.557781 384.093288 296.282935 541.37650 399.9241 406.41191 528.459639 321.1942 517.548790 566.089772 312.615737

Sample clustering

With all genes

Without X/Y genes

PCA on the normalized counts

With all factors

SPF vs GF

Order:

  • M, 8w
  • M, 52w
  • M, 104w
  • F, 8w
  • F, 52w
  • F, 104w

Gene co-expression analysis

Extract modules of co-expressed genes

  1. 19694
  2. 61
  1. 10291
  2. 61
pickSoftThreshold: will use block size 4347.
 pickSoftThreshold: calculating connectivity for given powers...
   ..working on genes 1 through 4347 of 10291
   ..working on genes 4348 through 8694 of 10291
   ..working on genes 8695 through 10291 of 10291
   Power SFT.R.sq  slope truncated.R.sq  mean.k. median.k. max.k.
1      1    0.227  1.310          0.952 1030.000  1.03e+03 1570.0
2      2    0.232 -0.842          0.911  328.000  3.12e+02  758.0
3      3    0.590 -1.550          0.954  132.000  1.15e+02  446.0
4      4    0.729 -1.880          0.974   61.300  4.74e+01  292.0
5      5    0.771 -2.080          0.972   31.700  2.12e+01  205.0
6      6    0.812 -2.130          0.981   17.800  1.01e+01  150.0
7      7    0.802 -2.220          0.974   10.600  5.05e+00  114.0
8      8    0.831 -2.190          0.983    6.630  2.61e+00   88.7
9      9    0.852 -2.140          0.988    4.330  1.40e+00   70.5
10    10    0.867 -2.110          0.991    2.920  7.74e-01   56.8
11    12    0.872 -2.060          0.983    1.440  2.55e-01   38.4
12    14    0.877 -2.010          0.977    0.779  9.07e-02   26.9
13    16    0.893 -1.940          0.982    0.449  3.40e-02   19.4
14    18    0.906 -1.860          0.985    0.273  1.33e-02   14.2
15    20    0.929 -1.780          0.993    0.173  5.31e-03   10.7
PowerSFT.R.sqslopetruncated.R.sqmean.k.median.k.max.k.
1 0.2273633 1.3069408 0.9522773 1032.21030621.031743e+031574.40467
2 0.2318240 -0.8419065 0.9109768 328.37500823.124206e+02 758.22758
3 0.5898524 -1.5468341 0.9541521 131.71067171.147058e+02 446.45478
4 0.7293365 -1.8760203 0.9740987 61.31571904.736691e+01 292.33277
5 0.7706350 -2.0775848 0.9724795 31.72701262.117472e+01 204.76919
6 0.8116126 -2.1271367 0.9807292 17.77317861.008260e+01 150.23963
7 0.8022879 -2.2224796 0.9735875 10.59251155.050971e+00 114.00564
8 0.8307511 -2.1879600 0.9827800 6.63451712.608895e+00 88.74112
9 0.8519550 -2.1423593 0.9884310 4.32820401.397797e+00 70.46121
10 0.8665930 -2.1052573 0.9914710 2.92122527.738615e-01 56.84323
12 0.8724663 -2.0611029 0.9830544 1.44476202.552006e-01 38.38129
14 0.8766131 -2.0101797 0.9767692 0.77888909.066889e-02 26.89092
16 0.8933757 -1.9356365 0.9817629 0.44880753.402853e-02 19.36386
18 0.9058564 -1.8638594 0.9845667 0.27268321.328237e-02 14.24265
20 0.9285263 -1.7822036 0.9931630 0.17299825.314736e-03 10.65525
Powermean.k.
6 6 17.7731786
7 7 10.5925115
8 8 6.6345171
9 9 4.3282040
1010 2.9212252
1112 1.4447620
1214 0.7788890
1316 0.4488075
1418 0.2726832
1520 0.1729982

Parameters for network construction

 Calculating module eigengenes block-wise from all genes
   Flagging genes and samples with too many missing values...
    ..step 1
 ..Working on block 1 .
    TOM calculation: adjacency..
    ..will use 4 parallel threads.
     Fraction of slow calculations: 0.000000
    ..connectivity..
    ..matrix multiplication (system BLAS)..
    ..normalization..
    ..done.
   ..saving TOM for block 1 into file norm_genes_TOM-block.1.RData
 ....clustering..
 ....detecting modules..
 ....calculating module eigengenes..
 ....checking kME in modules..
     ..removing 1 genes from module 1 because their KME is too low.
     ..removing 3 genes from module 9 because their KME is too low.
     ..removing 1 genes from module 12 because their KME is too low.
     ..removing 1 genes from module 13 because their KME is too low.
 ..merging modules that are too close..
     mergeCloseModules: Merging modules whose distance is less than 0.35
       Calculating new MEs...
 ME0  ME1  ME2  ME3  ME4  ME5  ME6  ME7  ME8  ME9 ME10 ME11 
4663 1239 1082  630  595  552  476  367  315  178  139   55 
11
  • 11 modules with between 55 and 1239 genes
  • 4663 genes not assigned to a module
5628
0
'grey'
1
'turquoise'
2
'blue'
3
'brown'
4
'yellow'
5
'green'
6
'red'
7
'black'
8
'pink'
9
'magenta'
10
'purple'
11
'greenyellow'

Relationship between modules and samples

Genes in modules

[1] "0: 147 - 3.15247694617199%"
[1] "1: 36 - 2.90556900726392%"
[1] "2: 53 - 4.89833641404806%"
[1] "3: 18 - 2.85714285714286%"
[1] "4: 13 - 2.18487394957983%"
[1] "5: 19 - 3.44202898550725%"
[1] "6: 9 - 1.89075630252101%"
[1] "7: 7 - 1.90735694822888%"
[1] "8: 9 - 2.85714285714286%"
[1] "9: 2 - 1.12359550561798%"
[1] "10: 1 - 0.719424460431655%"
[1] "11: 1 - 1.81818181818182%"

Heatmaps with Z-scores

Sinaplots of the Z-scores per groups

The mean of the Z-score over the samples in the group is plot for each gene

Median of counts over ages

Enrichment analysis in modules

[1] "0"
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
        category over_represented_pvalue under_represented_pvalue numDEInCat
9622  GO:0034641            1.930318e-34                        1       1689
11864 GO:0044237            1.819262e-33                        1       2515
9636  GO:0034660            1.511191e-32                        1        212
11881 GO:0044260            2.808359e-32                        1       2109
2983  GO:0006139            6.603118e-32                        1       1518
9532  GO:0034470            7.640556e-32                        1        169
      numInCat                                             term ontology
9622      5285     cellular nitrogen compound metabolic process       BP
11864     8386                       cellular metabolic process       BP
9636       405                          ncRNA metabolic process       BP
11881     6844         cellular macromolecule metabolic process       BP
2983      4672 nucleobase-containing compound metabolic process       BP
9532       301                                 ncRNA processing       BP
[1] "1"
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
        category over_represented_pvalue under_represented_pvalue numDEInCat
17661 GO:0090304            3.630391e-20                        1        450
11881 GO:0044260            1.095788e-18                        1        644
5710  GO:0016070            1.118447e-17                        1        406
2983  GO:0006139            9.921890e-17                        1        468
12852 GO:0046483            4.487238e-16                        1        471
11364 GO:0043170            1.928903e-15                        1        667
      numInCat                                             term ontology
17661     4172                   nucleic acid metabolic process       BP
11881     6844         cellular macromolecule metabolic process       BP
5710      3729                            RNA metabolic process       BP
2983      4672 nucleobase-containing compound metabolic process       BP
12852     4765                    heterocycle metabolic process       BP
11364     7455                  macromolecule metabolic process       BP
[1] "2"
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
        category over_represented_pvalue under_represented_pvalue numDEInCat
12259 GO:0045184            9.868354e-13                        1        181
11879 GO:0044257            1.106652e-12                        1         84
14123 GO:0051234            2.182943e-12                        1        345
5312  GO:0015031            2.460062e-12                        1        167
13314 GO:0048193            2.934615e-12                        1         40
3431  GO:0006810            4.433883e-12                        1        333
      numInCat                                  term ontology
12259     1765 establishment of protein localization       BP
11879      604    cellular protein catabolic process       BP
14123     4062         establishment of localization       BP
5312      1606                     protein transport       BP
13314      185               Golgi vesicle transport       BP
3431      3915                             transport       BP
[1] "3"
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
        category over_represented_pvalue under_represented_pvalue numDEInCat
17661 GO:0090304            8.971982e-19                        1        245
5710  GO:0016070            1.893249e-17                        1        223
14136 GO:0051252            2.138832e-15                        1        190
2983  GO:0006139            5.935538e-15                        1        251
12852 GO:0046483            7.973750e-15                        1        254
14083 GO:0051171            1.403879e-14                        1        210
      numInCat                                              term ontology
17661     4172                    nucleic acid metabolic process       BP
5710      3729                             RNA metabolic process       BP
14136     3046               regulation of RNA metabolic process       BP
2983      4672  nucleobase-containing compound metabolic process       BP
12852     4765                     heterocycle metabolic process       BP
14083     3601 regulation of nitrogen compound metabolic process       BP
[1] "4"
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
        category over_represented_pvalue under_represented_pvalue numDEInCat
11864 GO:0044237            3.108584e-18                        1        368
16807 GO:0071704            1.641624e-16                        1        377
11881 GO:0044260            1.908849e-16                        1        307
3934  GO:0008152            1.988270e-16                        1        390
11865 GO:0044238            2.158860e-16                        1        361
11364 GO:0043170            2.641483e-16                        1        328
      numInCat                                     term ontology
11864     8386               cellular metabolic process       BP
16807     8831      organic substance metabolic process       BP
11881     6844 cellular macromolecule metabolic process       BP
3934      9217                        metabolic process       BP
11865     8362                primary metabolic process       BP
11364     7455          macromolecule metabolic process       BP
[1] "5"
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
        category over_represented_pvalue under_represented_pvalue numDEInCat
3553  GO:0007005            4.130830e-20                        1         70
3934  GO:0008152            1.159491e-17                        1        342
14836 GO:0055114            5.773753e-16                        1         76
11864 GO:0044237            7.659119e-16                        1        309
3431  GO:0006810            8.277589e-16                        1        167
14123 GO:0051234            1.090689e-14                        1        167
      numInCat                          term ontology
3553       611    mitochondrion organization       BP
3934      9217             metabolic process       BP
14836      836   oxidation-reduction process       BP
11864     8386    cellular metabolic process       BP
3431      3915                     transport       BP
14123     4062 establishment of localization       BP
[1] "6"
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
        category over_represented_pvalue under_represented_pvalue numDEInCat
12058 GO:0044710            6.653859e-23                        1        174
4336  GO:0009056            3.116046e-13                        1         88
12060 GO:0044712            4.296236e-13                        1         52
18752 GO:1901575            4.041497e-12                        1         83
3934  GO:0008152            1.327891e-11                        1        306
2882  GO:0005975            1.537646e-11                        1         41
      numInCat                                term ontology
12058     3280   single-organism metabolic process       BP
4336      1533                   catabolic process       BP
12060      635   single-organism catabolic process       BP
18752     1478 organic substance catabolic process       BP
3934      9217                   metabolic process       BP
2882       489      carbohydrate metabolic process       BP
[1] "7"
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
        category over_represented_pvalue under_represented_pvalue numDEInCat
13833 GO:0050794            2.673494e-09                        1        248
6626  GO:0019222            1.600242e-08                        1        167
5710  GO:0016070            2.997303e-08                        1        129
7965  GO:0031323            3.075312e-08                        1        159
13829 GO:0050789            4.088212e-08                        1        253
11881 GO:0044260            4.439255e-08                        1        201
      numInCat                                     term ontology
13833     8793           regulation of cellular process       BP
6626      5208          regulation of metabolic process       BP
5710      3729                    RNA metabolic process       BP
7965      4897 regulation of cellular metabolic process       BP
13829     9278         regulation of biological process       BP
11881     6844 cellular macromolecule metabolic process       BP
[1] "8"
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
        category over_represented_pvalue under_represented_pvalue numDEInCat
3523  GO:0006955            2.985898e-25                        1         70
1046  GO:0002376            3.655761e-25                        1        100
3520  GO:0006952            1.989081e-22                        1         72
12206 GO:0045087            7.941872e-22                        1         47
11393 GO:0043207            2.651971e-17                        1         49
14391 GO:0051707            2.651971e-17                        1         49
      numInCat                                 term ontology
3523       982                      immune response       BP
1046      1975                immune system process       BP
3520      1156                     defense response       BP
12206      500               innate immune response       BP
11393      686 response to external biotic stimulus       BP
14391      686           response to other organism       BP
[1] "9"
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
        category over_represented_pvalue under_represented_pvalue numDEInCat
1211  GO:0002682            2.613030e-07                0.9999999         30
1213  GO:0002684            2.877916e-07                0.9999999         23
952   GO:0002221            1.182405e-06                0.9999999          9
7990  GO:0031349            1.266030e-06                0.9999998         13
1267  GO:0002758            1.395896e-06                0.9999999          9
13445 GO:0048518            1.634916e-06                0.9999992         77
      numInCat                                                  term ontology
1211      1088                   regulation of immune system process       BP
1213       676          positive regulation of immune system process       BP
952        104        pattern recognition receptor signaling pathway       BP
7990       252               positive regulation of defense response       BP
1267       106 innate immune response-activating signal transduction       BP
13445     4751             positive regulation of biological process       BP
[1] "10"
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
[1] category                 over_represented_pvalue  under_represented_pvalue
[4] numDEInCat               numInCat                 term                    
[7] ontology                
<0 rows> (or 0-length row.names)
[1] "11"
Warning message in grep(txdbPattern, installedPackages):
“argument 'pattern' has length > 1 and only the first element will be used”Warning message in pcls(G):
“initial point very close to some inequality constraints”Fetching GO annotations...
For 1495 genes, we could not find any categories. These genes will be excluded.
To force their use, please run with use_genes_without_cat=TRUE (see documentation).
This was the default behavior for version 1.15.1 and earlier.
Calculating the p-values...
'select()' returned 1:1 mapping between keys and columns
[1] category                 over_represented_pvalue  under_represented_pvalue
[4] numDEInCat               numInCat                 term                    
[7] ontology                
<0 rows> (or 0-length row.names)

Network visualization

TOM calculation: adjacency..
..will use 4 parallel threads.
 Fraction of slow calculations: 0.000000
..connectivity..
..matrix multiplication (system BLAS)..
..normalization..
..done.
0610007P14Rik0610009B22Rik0610009O20Rik0610010K14Rik0610030E20Rik0610037L13RikFmc1Card191110008F13Rik1110008P14RikZswim7Zswim8ZufspZwintZxdaZyg11bZyxZzef1Zzz3Hikeshi
0610007P14Rik11000000100100000000
0610009B22Rik11100000000100000001
0610009O20Rik01100001000100100000
0610010K14Rik00010001001000011001
0610030E20Rik00001100100111010110
0610037L13Rik00001100000110000110
0610007P14Rik
'5'
0610009B22Rik
'2'
0610009O20Rik
'7'
0610010K14Rik
'5'
0610030E20Rik
'1'
0610037L13Rik
'3'
5
'green'
2
'blue'
7
'black'
5
'green'
1
'turquoise'
3
'brown'